Detection of target nucleic acid sequences using fluorescence resonance energy transfer

Abstract

A method for identifying a plurality of target nucleic acid molecules in a sample. The method provides a plurality of oligonucleotide probe sets. Each set comprises a first and a second probe, each having a target-specific portion and a tunable portion with an acceptor or a donor group. The first probe further comprises an endcapped hairpin. A reaction comprises a denaturation and hybridization cycle. Under the hybridization, the set of probes hybridize in a base-specific manner to their respective target nucleotide sequences, and ligate to one another to form a ligation product. Under conditions that permit hybridization of the tunable portions of the ligation product to one another, an internally hybridized ligation product formed, which allows the detection of the fluorescence resonance energy transfer (FRET). A method comprising PCR amplification is also disclosed.

Description

[0001]This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61 / 043,282, filed Apr. 8, 2008, which is hereby incorporated by reference in its entirety.[0002]This invention was made with government support under Public Health Service grant AI062579-03 from the National Institute of Allergy and Infectious Diseases and Grant No. CA65930-08 from the National Cancer Institute. The government has certain rights in this invention.FIELD OF THE INVENTION[0003]The present invention relates to the detection of target nucleic acid sequences using fluorescence resonance energy transfer (FRET).BACKGROUND OF THE INVENTION[0004]Advances in sequencing technology have led to the proliferation of whole genomic sequence data beginning with the publication of the human genome. The availability of this sequence information has allowed for the design and development of DNA based diagnostic tests for several genetic diseases, e.g. various cancers, cystic fibrosis, etc. In the m...

AI Technical Summary

Benefits of technology

[0021]The primary advantage of the present invention is the utilization of oligonucleotide probes bearing tunable portions that include a tunable endcapped hairpin, which allows for the donor and acceptor fluorophores to undergo FRET at predefined melting temperatures. Since the melting temperature (“Tm”) of the ligation product hairpin is controlled by the sequence of the approximately 4 to 6 perfectly complementary bases adjacent to, as well as the structure of the endcapped sequence, it is independent of the LDR probe sequences as well as independent of the LDR product sequence length. Further, it is distinguished by having a tight melting profile. Thus, the endcapped design overcomes the deficiencies of spFRET-LDR probes. The tunable endcapped hairpin approach allows for design of a standardized set of sequences which may be appended to LDR probes, giving reliable and predictable Tm values that are easily distinguishable in a multiplexed format, independent of the target sequence being probed. When coupled to LDR assays, the probes of the present invention enable multiplexing of between 48-60 signals. Assays can be performed on standard real time PCR instrumentation, such as the ABI 7500 or the Cepheid GeneXpert system, which enables both thermal cycling for the LDR as well as readout of the signals. When used for real time assays analogous to Taqman® probes, the oligonucleotide probes of the present invention allow for multiplexing of up to 18 targets simultaneously.

is the utilization of oligonucleotide probes bearing tunable portions that include a tunable endcapped hairpin, which allows for the donor and acceptor fluorophores to undergo FRET at predefined melting temperatures. Since the melting temperature (“Tm”) of the ligation product hairpin is controlled by the sequence of the approximately 4 to 6 perfectly complementary bases adjacent to, as well as the structure of the endcapped sequence, it is independent of the LDR probe sequences as well as independent of the LDR product sequence length. Further, it is distinguished by having a tight melting profile. Thus, the endcapped design overcomes the deficiencies of spFRET-LDR probes. The tunable endcapped hairpin approach allows for design of a standardized set of sequences which may be appended to LDR probes, giving reliable and predictable Tm values that are easily distinguishable in a multiplexed format, independent of the target sequence being probed. When coupled to LDR assays, the probes of the present invention enable multiplexing of between 48-60 signals. Assays can be performed on standard real time PCR instrumentation, such as the ABI 7500 or the Cepheid GeneXpert system, which enables both thermal cycling for the LDR as well as readout of the signals. When used for real time assays analogous to Taqman® probes, the oligonucleotide probes of the present invention allow for multiplexing of up to 18 targets simultaneously.

Problems solved by technology

A disadvantage of these methods, however, is their lack of multiplexing capability as they are limited to simultaneous use of 4-6 dyes, of which one is an internal control.

However, both the electrophoresis and universal array methods for separating and detecting the ligation products require an additional step that must be performed after the LDR reaction using additional equipment.

spFRET-LDR is not ideal for obtaining a multiplexed readout signal, wherein the same donor and acceptor molecules are used to provide readouts at different temperatures.

For these reasons, it is difficult to design a set of LDR probes having the same acceptor and donor groups to different targets, where two or more LDR product signals may be readily distinguished through their different Tm values.

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